Genetic mutations in genes encoding presenilins (PS1/PS2) are linked to the majority of familial Alzheimer's disease (FAD, AD) cases. PS1 and PS2 (PS) play a critical role in proteolysis of beta-amyloid precursor protein (APP) to generate beta-amyloid (Abeta), a peptide important in AD pathogenesis. These multi-transmembrane proteins accumulate as endoproteolyzed heterodimers of N- and C-terminal fragments and associate with other membrane proteins (e.g., nicastrin, APH-1 and PEN-2) to form high molecular weight complexes that possess gamma-secretase activity cleaving type I membrane proteins including betaAPP and Notch-1. However, little is known about the biology of nicastrin, APH1 and PEN2, and the nature of their involvement in betaAPP/Notch processing. Nevertheless, numerous reports have assigned additional physiological functions to presenilins, including roles in calcium homeostasis, skeletal development, neurite outgrowth, apoptosis, synaptic plasticity and tumorigenesis. Recent evidence from several groups including ours suggest a role for PS1 in regulating intracellular trafficking/maturation, within the secretory pathways of select proteins including APP, nicastrin, TrkB and a PS1-binding protein (ICAM-5/telecephalin). In preliminary studies using a cell-free vesicle trafficking reconstitution system, we demonstrated that PS1 regulates the biogenesis of APP-containing vesicles from the trans-Golgi Network (TGN) and the endoplasmic reticulum (ER). PS1 deficiency or the expression of loss-of-function variants leads to robust vesicle formation, concomitant with increased maturation and/or cell surface accumulation of APP. In contrast, biogenesis of APP-containing vesicles is impaired in FAD-linked PS1 mutant cells, resulting in reduced APP delivery to the cell surface. Moreover, diminution of surface APP is profound at axonal terminals in neurons expressing a PS1 FAD variant. Our results also suggested that recruitment of some cytosolic trafficking factors to donor organelles/membranes may be affected by loss of PS1 functions, and that gain of function PS1 FAD mutations may alleviate the intrinsic requirement of ATP and temperature for Abeta generation, implicating a chaperone-like function for PS1. These results suggest that PS1 regulation of APP trafficking may represent an alternative mechanism by which FAD-linked PS1 variants modulate APP processing along a pathologic, amyloidogenic pathway. In this proposal, Aim 1 addresses the cellular mechanism underlying PS1 regulation of APP trafficking from the TGN and the ER, by determining roles of PS1 in recruiting cytosolic factors (e.g. phosholipases and small GTP-binding proteins) and in affecting ATP- and/or temperature dependent vesicle biogenesis. Aim 2 proposes to determine whether PS1 regulates intracellular trafficking of other membrane proteins that are components of the y-secretase complex (e.g., nicastrin, APH1 and PEN2) and how these molecules influence APP processing/trafficking. Aim 3 proposes to assess physiological implications of PS1 regulated axonal transport of APP in neuronal functions such as neurite outgrowth and synapse formation.